The monoclonal antibody 265-3K1 recognizes human leukocyte elastase. Leukocyte elastase, a major serine proteinase in man, is predominantly present in the azurophilic granules of neutrophils and monocytes. Elastase has a broad range of extracellular matrix substrates including elastin, proteoglycans, collagen and fibronectin. The action of elastase is controlled by serine proteinase inhibitors. Elastase, when released during inflammation, is rapidly bound by its two main inhibitors, alpha1-PI and alpha2-macroglobuline to form elastase-inhibitor complexes. In addition mucosa secretions may contain the locally secreted elastase inhibitors elafin/SKALP and SLPI. When secreted at sites of inflammation elastase can cause severe tissue damage. An important role has been suggested for human elastase in various inflammatory disorders, including pulmonary emphysema, sepsis, arthritis, nephritis and certain skin diseases. Elastase induces the production of IL-8 in human bronchial epithelial, a proces that occurs in part through TLR4.
Flow cytometry, Immuno assays, Immuno fluorescence, Paraffin sections, Western blot
IA: the antibody can be used as capture antibody. Antibody 265-3K1 was unique in that the limit of detection as a capture antibody increased from 625 nM (18.8 ng/ml) for free HNE to 80 nM for HNE–AAT complex, indicating that covalent complex formation between HNE and AAT increases antibody affinity or increases exposure of recognised epitope in HNE for 265-3K1 (Ref.1).
FC: cells were fixed with ADG fixation buffer A for 15 min at room temperature. Cells were washed and incubated with 10 μg of monoclonal anti-HNE antibody diluted in ADG permeabilisation buffer B (Ref.1).
IF: tissues were fixed in paraformaldehyde (PFA) 3.7%, embedded in paraffin and sectioned at 6 mm. The sections were deparaffinized in toluene and hydrated in graded series of ethanol (Ref.2).
Dilutions to be used depend on detection system applied. It is recommended that users test the reagent and determine their own optimal dilutions. The typical starting working dilution is 1:50.